CN103555783A - Method for preparing phosphatidyserine - Google Patents
Method for preparing phosphatidyserine Download PDFInfo
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- CN103555783A CN103555783A CN201310508296.3A CN201310508296A CN103555783A CN 103555783 A CN103555783 A CN 103555783A CN 201310508296 A CN201310508296 A CN 201310508296A CN 103555783 A CN103555783 A CN 103555783A
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Abstract
The invention relates to a method for preparing phosphatidyserine. The method for preparing phosphatidyserine comprises the following steps of mixing phosphatidylcholine or natural phospholipid containing phosphatidylcholine with tween-80, L-serine, calcium chloride, buffer solution and phospholipase D, and stirring and reacting the above materials at 35-45 DEG C for 8-10h; separating out the crude products from reaction and floating, carrying out centrifugal separation on the aqueous phase and crude products, washing the crude products subjected to separation and carrying out centrifugal filtration; adding the crude products subjected to centrifugal filtration into absolute ethyl alcohol for immersing, stirring, and carrying out centrifugal filtration; and carrying out vacuum drying on the products subjected to centrifugal filtration to obtain the phosphatidyserine. The invention aims to provide a new method for preparing phosphatidyserine to solve the problems that in the prior art, the byproduct of phosphatidic acid is large in amount, the conversion rate is unsatisfactory and the product cost is high.
Description
Technical field
The present invention relates to a kind of method of preparing stable powdered phosphatidyl serine.
Background technology
Phosphatide is the abbreviation of glycerophospholipids, and the eubolism of biomembranous biological activity and body is had to important regulatory function.Usually said phosphatide refers to the fatty substance that contains phosphoric, it is the mixture of multiple phosphatide, and composition is mainly phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositols (PI), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidyl glycerol (PG) etc.
Phosphatidylserine is a kind of unique phosphatide that can regulating cell film key protein function, many cellular process are had to important regulating effect, its major function is to improve brain cell activity, to treatment encephalatrophy, prevention senile dementia, the brain function that improves the elderly has good curative effect; Repair brain damage simultaneously, treat the effect that childhood hyperkinetic syndrome has highly significant; Can promote in addition recovery, balance mood, the alleviate depression of brain fag also to have certain curative effect.
The preparation method of phosphatidylserine mainly contains extraction method and enzyme transforming process.Wherein extraction method is mainly to extract PS from the Yelkin TTS of vegetable cell, animal, PS content in plant is less, so be extracted as master in the Yelkin TTS of extraction method with animal, from animal, extracting is mainly brain and the internal organ of animal, and external most of animal brain of take the poultry such as ox, sheep, rabbit, horse, donkey is extracted PS as raw material at present.In recent years, due to mad cow disease etc. Animal diseases, utilize zooblast to extract the method for PS, people's suspection is received in the security of the product of its extraction, now in eliminating edge.
Enzyme transforming process is mainly to take natural Yelkin TTS as matrix, adds Serine, under the effect of Phospholipase D, generates phosphatidylserine.Scientific literature and patent report the method that is phosphatidylserine by Phospholipase D catalysis by phosphatidylcholine, these method major parts are the two-phase systems by water/organic solvent, but two-phase system organic solvent usage quantity is large, be difficult to remove, increased reaction cost simultaneously.Have investigator's single water react that begins one's study, patent EP1310563 has described the method for preparing PS in the water that does not use washing composition and/or calcium salt, and the method need to will comprise phosphatide in water, the starting mixt homogenate of hydroxyl receptor and Phospholipid hydrolase.The method of preparing PS in pure aquatic system that patent JP2002051794 and DE102004002053 announce also will be by the special equipment that homogenizes.Patent CN101230365 adopts single water, but water consumption is large, maximum has been used 10 times of response matrix, and the 4-6 that the consumption of its Serine is response matrix doubly measures, after reaction finishes, used organic solvent extraction, when having introduced organic solvent, also increased reaction cost simultaneously.Patent CN102676600 has described all reacting in phase medium γ-valerolactone, 2-methyltetrahydrofuran or its mixture, has increased production cost.And in patent CN101230365 long reaction time, temperature of reaction is high, causes in reaction product PA content high, PS low conversion rate, reaction product poor quality.
Summary of the invention
The object of this invention is to provide a kind of new method of preparing phosphatidylserine, more to solve the by product phosphatidic acid that prior art exists, transformation efficiency is undesirable, the problem that production cost is high.
The present invention adopts following solution:
A method of preparing phosphatidylserine, mainly comprises the following steps:
(1) phosphatidylcholine or the natural phospholipid that contains phosphatidylcholine are mixed with tween-80, Serine, calcium chloride, buffered soln, Phospholipase D, at 35-45 ℃ of stirring reaction 8-10h;
Wherein, tween-80 is the 0.1-0.5% of natural phospholipid quality, Serine is 2-3 times of natural phospholipid quality, the pH value of buffered soln is 5.1-5.5, the add-on of buffered soln is 5-6 times of natural phospholipid quality, the add-on of Phospholipase D is every g natural phospholipid 20-50U, and the ratio of calcium chloride and buffered soln is 1.5-1.8g/100mL;
(2) reaction finishes rear crude reaction and separates out and float, centrifugation water and crude reaction, and the crude reaction that separation is obtained washes with water, and centrifuging;
(3) crude reaction after centrifuging adds soaked in absolute ethyl alcohol, stir, then centrifuging;
(4) to the vacuum-drying of centrifuging after product, obtain phosphatidylserine product.
Based on above-mentioned general planning, the present invention also does following optimization and limits and improve:
Configuration selection acetic acid-sodium-acetate system or the Sodium phosphate dibasic-Phosphoric Acid of above-mentioned buffered soln.
The 2-4 that the middle dehydrated alcohol add-on of above-mentioned steps (3) is now crude reaction quality doubly, stirs 1-2h after soaked in absolute ethyl alcohol 2-4h, then centrifuging.
In above-mentioned steps (2) water washing doubly measure into the 0.5-1 of crude reaction now doubly.
In above-mentioned steps (4), vacuum drying parameter is 50 ± 2 ℃ of temperature, vacuum tightness 0.08 ± 0.005Mpa.
Natural phospholipid wide material sources of the present invention, preferably soybean phospholipid, sunflower seeds phosphatide, egg phospholipids.Wherein phosphatidylcholine content all can react at 10-95%, the high-content phosphatidylcholine after more preferably purified.
Phospholipase D of the present invention can derive from plant and microorganism.Plant origin comprises Chinese cabbage, spinach, wild cabbage, Radix Dauci Sativae etc., microbe-derived streptomycete, intestinal bacteria, the Salmonella etc. of comprising, the streptomycete product enzyme that preferred enzyme is lived higher.
Beneficial effect of the present invention and feature:
The present invention utilizes the emulsifying effect of tween-80 to make response matrix form glue, and without the special equipment that homogenizes, shortened the reaction times has increased transformation efficiency simultaneously.
In whole technological process, except ethanol, without other organic solvent, bring into, guaranteed the green health of product.Through evidence reaction product, by soaked in absolute ethyl alcohol, can destroy the Phospholipase D of bringing in product active, make product stability better, the solvable oil soluble composition of taking off in reaction product of while dehydrated alcohol, make the mobility of the finished product, taste, color and luster is better, can be used for better preparing capsule or other protective foodss containing phosphatidylserine.
Whole technical process is simple, and speed of response is fast, and the cycle is short, it is little to invest, and cost is low.Reaction product yield is high, and purity is high, quality better.
Embodiment
Below in conjunction with embodiment, the present invention is elaborated.
Embodiment 1
In retort, add the soybean phospholipid that 20g phosphatidylcholine content is 50.98%, Serine 40g, tween-80 0.02g, calcium chloride 1.6g, buffered soln is selected sodium-acetate-acetate system, pH value 5.5, buffered soln add-on 100mL, Phospholipase D enzyme is lived as 5000U/g, and add-on is 100mg.Stir and keep 37 ℃ of temperature of reaction, reaction times 8h, detect transformation efficiency, centrifuging, centrifugal rear filter residue adds water 20mL washing three times, and the crude reaction of centrifuging, crude reaction adds dehydrated alcohol 100mL, after soaking 2h, stir 1h, centrifuging, vacuum-drying obtains the powder lecithin that 17.28g PS content is 56.48%.
Embodiment 2
In retort, add the sunflower seeds phosphatide that 50g phosphatidylcholine content is 80.68%, Serine 150, tween-80 0.2g, calcium chloride 6.0g, buffered soln is selected sodium-acetate-acetate system, pH value 5.3, buffered soln add-on 400mL, Phospholipase D enzyme is lived as 5000U/g, and add-on is 400mg.Stir and keep 40 ℃ of temperature of reaction, reaction times 10h, detect transformation efficiency, centrifuging, centrifugal rear filter residue adds water 50mL washing three times, and the crude reaction of centrifuging, crude reaction adds dehydrated alcohol 400mL, after soaking 2h, stir 1h, centrifuging, vacuum-drying obtains the powder lecithin that 44.88g PS content is 87.75%.
Embodiment 3
In retort, add the soybean phospholipid that 50g phosphatidylcholine content is 21.62%, Serine 100g, tween-80 0.25g, calcium chloride 6.0g, buffered soln is selected Sodium phosphate dibasic-Phosphoric Acid, pH value 5.1, buffered soln add-on 300mL, Phospholipase D enzyme is lived as 5000U/g, and add-on is 220mg.Stir and keep 37 ℃ of temperature of reaction, reaction times 10h, detect transformation efficiency, centrifuging, centrifugal rear filter residue adds water 50mL washing three times, and the crude reaction of centrifuging, crude reaction adds dehydrated alcohol 300mL, after soaking 2h, stir 1h, centrifuging, vacuum-drying obtains the powder lecithin that 33.89g PS content is 30.27%.
Embodiment 4
In retort, add the egg phospholipids that 50g phosphatidylcholine content is 30.89%, Serine 120g, tween-80 0.15g, calcium chloride 8.0, buffered soln is selected Sodium phosphate dibasic-Phosphoric Acid, pH value 5.3, buffered soln add-on 500mL, Phospholipase D enzyme is lived as 5000U/g, and add-on is 400mg.Stir and keep 37 ℃ of temperature of reaction, reaction times 8h, detect transformation efficiency, centrifuging, centrifugal rear filter residue adds water 50mL washing three times, and the crude reaction of centrifuging, crude reaction adds dehydrated alcohol 400mL, after soaking 2h, stir 1h, centrifuging, vacuum-drying obtains the powder lecithin that 41.27g PS content is 35.78%.
In each embodiment, reaction product component content and transformation efficiency see the following form 1.
Claims (7)
1. a method of preparing phosphatidylserine, mainly comprises the following steps:
(1) phosphatidylcholine or the natural phospholipid that contains phosphatidylcholine are mixed with tween-80, Serine, calcium chloride, buffered soln, Phospholipase D, at 35-45 ℃ of stirring reaction 8-10h;
Wherein, tween-80 is the 0.1-0.5% of natural phospholipid quality, Serine is 2-3 times of natural phospholipid quality, the pH value of buffered soln is 5.1-5.5, the add-on of buffered soln is 5-6 times of natural phospholipid quality, the add-on of Phospholipase D is every g natural phospholipid 20-50U, and the ratio of calcium chloride and buffered soln is 1.5-1.8g/100mL;
(2) reaction finishes rear crude reaction and separates out and float, centrifugation water and crude reaction, and the crude reaction that separation is obtained washes with water, and centrifuging;
(3) crude reaction after centrifuging adds soaked in absolute ethyl alcohol, stir, then centrifuging;
(4) to the vacuum-drying of centrifuging after product, obtain phosphatidylserine product.
2. the method for preparing phosphatidylserine according to claim 1, is characterized in that:
Configuration selection acetic acid-sodium-acetate system or the Sodium phosphate dibasic-Phosphoric Acid of described buffered soln.
3. the method for preparing phosphatidylserine according to claim 2, is characterized in that:
The 2-4 that the middle dehydrated alcohol add-on of step (3) is now crude reaction quality doubly, stirs 1-2h after soaked in absolute ethyl alcohol 2-4h, then centrifuging.
4. the method for preparing phosphatidylserine according to claim 3, is characterized in that: in step (2) water washing doubly measure into the 0.5-1 of crude reaction now doubly.
5. the method for preparing phosphatidylserine according to claim 4, is characterized in that: in step (4), vacuum drying parameter is 50 ± 2 ℃ of temperature, vacuum tightness 0.08 ± 0.005Mpa.
6. according to the arbitrary described method of preparing phosphatidylserine of claim 1 to 5, it is characterized in that: described natural phospholipid derives from soybean phospholipid, sunflower seeds phosphatide or egg phospholipids.
7. according to the arbitrary described method of preparing phosphatidylserine of claim 1 to 5, it is characterized in that: described Phospholipase D derives from plant and microorganism, wherein plant origin comprises Chinese cabbage, spinach, wild cabbage and Radix Dauci Sativae, microbe-derived streptomycete, intestinal bacteria and the Salmonella of comprising.
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105087700A (en) * | 2015-08-31 | 2015-11-25 | 上海裕兰生物科技有限公司 | Preparation method of high-content phosphatidylserine |
| CN105296560A (en) * | 2015-12-07 | 2016-02-03 | 杏辉天力（杭州）药业有限公司 | Preparation method of phosphatidylserine |
| CN105567755A (en) * | 2016-01-28 | 2016-05-11 | 成都显诚生物工程有限公司 | Preparation method of phosphatidylserine |
| CN107266493A (en) * | 2017-06-12 | 2017-10-20 | 芜湖福民生物药业股份有限公司 | The preparation method of phosphatidylserine |
| CN107266492A (en) * | 2017-06-12 | 2017-10-20 | 芜湖福民生物药业股份有限公司 | The extracting method of phosphatidylserine |
| CN107334008A (en) * | 2017-06-14 | 2017-11-10 | 芜湖福民生物药业股份有限公司 | Fruit beverage containing phosphatidylserine and preparation method thereof |
| CN108103115A (en) * | 2018-03-02 | 2018-06-01 | 江苏中酶生物科技有限公司 | A kind of method that micro- water mutually prepares phosphatidic acid |
| CN109486790A (en) * | 2018-12-10 | 2019-03-19 | 南通励成生物工程有限公司 | A method of phosphatidyl serine is prepared with phospholipase D conversion |
| CN109628517A (en) * | 2018-12-27 | 2019-04-16 | 南通励成生物工程有限公司 | A kind of method that ultrasound-assisted enzymolysis prepares phosphatidylserine |
| CN112352051A (en) * | 2019-12-31 | 2021-02-09 | 邦泰生物工程(深圳)有限公司 | Method for improving phospholipase D (phospholipase D) translipidation activity and method for producing phosphatidylserine by using phospholipase D translipidation activity |
| CN112384630A (en) * | 2019-12-31 | 2021-02-19 | 邦泰生物工程(深圳)有限公司 | Anti-viscosity method for catalytic production of phosphatidylserine enzyme and method for producing phosphatidylserine by using same |
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| EP1310563A1 (en) * | 2000-08-09 | 2003-05-14 | Kabushiki Kaisha Yakult Honsha | Process for the production of phospholipids |
| CN101230365A (en) * | 2008-01-08 | 2008-07-30 | 西安皓天生物工程技术有限责任公司 | Method for preparing powdered phosphatidyl serine |
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2013
- 2013-10-24 CN CN201310508296.3A patent/CN103555783B/en active Active
Patent Citations (2)
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| EP1310563A1 (en) * | 2000-08-09 | 2003-05-14 | Kabushiki Kaisha Yakult Honsha | Process for the production of phospholipids |
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Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105087700A (en) * | 2015-08-31 | 2015-11-25 | 上海裕兰生物科技有限公司 | Preparation method of high-content phosphatidylserine |
| CN105296560A (en) * | 2015-12-07 | 2016-02-03 | 杏辉天力（杭州）药业有限公司 | Preparation method of phosphatidylserine |
| CN105567755A (en) * | 2016-01-28 | 2016-05-11 | 成都显诚生物工程有限公司 | Preparation method of phosphatidylserine |
| CN107266493A (en) * | 2017-06-12 | 2017-10-20 | 芜湖福民生物药业股份有限公司 | The preparation method of phosphatidylserine |
| CN107266492A (en) * | 2017-06-12 | 2017-10-20 | 芜湖福民生物药业股份有限公司 | The extracting method of phosphatidylserine |
| CN107334008A (en) * | 2017-06-14 | 2017-11-10 | 芜湖福民生物药业股份有限公司 | Fruit beverage containing phosphatidylserine and preparation method thereof |
| CN108103115A (en) * | 2018-03-02 | 2018-06-01 | 江苏中酶生物科技有限公司 | A kind of method that micro- water mutually prepares phosphatidic acid |
| CN109486790A (en) * | 2018-12-10 | 2019-03-19 | 南通励成生物工程有限公司 | A method of phosphatidyl serine is prepared with phospholipase D conversion |
| CN109628517A (en) * | 2018-12-27 | 2019-04-16 | 南通励成生物工程有限公司 | A kind of method that ultrasound-assisted enzymolysis prepares phosphatidylserine |
| WO2020133126A1 (en) * | 2018-12-27 | 2020-07-02 | 南通励成生物工程有限公司 | Method for preparing phosphatidylserine by ultrasonic assisted enzymolysis |
| US11390895B2 (en) * | 2018-12-27 | 2022-07-19 | Nantong Licheng Biological Engineering Co. Ltd | Method for preparing phosphatidylserine by ultrasonic-assisted enzymatic hydrolysis |
| CN112352051A (en) * | 2019-12-31 | 2021-02-09 | 邦泰生物工程(深圳)有限公司 | Method for improving phospholipase D (phospholipase D) translipidation activity and method for producing phosphatidylserine by using phospholipase D translipidation activity |
| CN112384630A (en) * | 2019-12-31 | 2021-02-19 | 邦泰生物工程(深圳)有限公司 | Anti-viscosity method for catalytic production of phosphatidylserine enzyme and method for producing phosphatidylserine by using same |
| WO2021134440A1 (en) * | 2019-12-31 | 2021-07-08 | 邦泰生物工程(深圳)有限公司 | Method for resisting viscosity during enzyme-catalyzed production of phosphatidylserine and method for producing phosphatidylserine using same |
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